Project Summary/Abstract The long-term goal of this application is to understand the molecular mechanisms a cell uses to maintain genome stability. Genome instability is a hallmark of cancer, and defects in genome maintenance genes predispose individuals to cancer. Such defects also cause neurogenerative disorders and developmental problems. Recently, an siRNA screen was completed in mammalian cells to identify genes which, when lost, lead to increased phosphorylation of 3H2AX, a marker for DNA double-strand breaks (DSBs). Enriched among the hits are genes with established roles in RNA metabolism and transcription, and it was shown that for many of these hits, H2AX phosphorylation is dependent on the formation of RNA-DNA hybrids, structures also known as R-loops. It is not clear how R-loops lead to DNA damage in cells, and the basic biological mechanisms that prevent the accumulation of these structures are not known. In the first aim of this application, the molecular mechanisms by which R-loops lead to DNA damage will be examined, using several of the genes identified in the siRNA screen to probe these events. In addition, ChIP-Seq approaches will be used to determine where DNA damage occurs in the genome, and to assess whether the sites of DNA damage are also the same sites where R-loops form. In the second aim, the molecular function of one gene identified in the screen, a DExxQ- type helicase that leads to increased R-loop formation when lost, will be studied. It is hypothesized that this helicase plays a direct role in processing R-loops thereby protecting cells against DNA damage and genome instability. Biochemical and genetic approaches will be used to determine the role of the helicase domain in preventing DNA damage and to assess the types of structures on which the helicase acts. These studies will help us elucidate the function of a novel genome maintenance gene and provide a better understanding of how defects in RNA metabolism can affect genome stability.